The invention relates to DNA molecules encoding a canine IgA. The invention comprises the DNA molecules, proteins encoded by the DNA molecules, antibodies to the proteins, cells transformed by the DNA molecules, assays employing the transformed cells, compounds identified by the assays and kits containing the DNA molecules or derivatives thereof.
Immunoglobulin (Ig) proteins consist of two identical light (L) chains and two identical heavy (H)chains. Both Ig L and H chains contain an amino-terminal variable region of approximately 110 amino acids that forms the antigen binding domain. The carboxy terminal constant (C) region domains of each chain is defined by two isotypes of IgL chain ( kappa and lambda) and multiple isotypes of IgH chains (mu, delta, gamma, epsilon and alpha which define IgM, IgD, IgG, IgE, and IgA, respectively). The IgH chain C regions contain the effector functions common to antibodies of a given isotype.
Substantial variations in the quantities of specific IgH chain isotypes are observed when different tissue fluids are analyzed. For instance, IgA is the primary Ig isotype in mucosal fluids, but is found at low levels in serum. The preponderance of IgA found at mucosal sites reflects the critical role IgA provides as a first line of defense against pathogens invading epithelial surfaces.
IgA is the principal immunoglobulin of mucosal surfaces where it is secreted as a polymeric antibody complex that contains J chain and secretory component. To fully understand antibody mediated immune responses at mucosal surfaces in a specific species, a knowledge of the Ig.alpha. constant region gene from that species is required.
Substantial variation in Ig.alpha. C regions gene copy number among species has been reported. For instance, functional Ig.alpha. gene copy number differs widely from mouse (one) and human (two) to rabbit. Our studies indicates that only a single Ig.alpha. gene is present in the canine genome. Unlike other immunoglobulin genes, the hinge region of Ig.alpha. is not encoded by a separate exon, but rather is fused to the 5' end of the CH2 domain. This organization of hinge and CH2 is conserved in the canine Ig.alpha. gene. The length of the Ig.alpha. hinge region has been shown to vary. For instance, the human Ig.alpha.1 gene has a hinge region of 18 amino acids while the human Ig.alpha.2 gene hinge region is only 5 amino acids long. The canine Ig.alpha. gene hinge region is 10 amino acids long, identical in length to the mouse Ig.alpha. hinge region.
Studies have suggested that the primary interactions between IgA and secretory component reside in the CH2 and CH3 domains of Ig.alpha.. Secretory component is covalently linked to IgA through disulfide bonds. Cys 311 of the human Iga CH2 domain is responsible for this linkage. Covalent linkage of canine Ig.alpha. to secretory component may also occur at this position because this cysteine residue is conserved in the canine Ig.alpha. chain.
For therapeutic purposes, it may be desirable to generate antibodies against the IgA of the target species in order to maximize the affinity of the anti-IgA antibodies. In addition, screening assays aimed at the identification of small molecules which alter IgA mediated responses in the dog can be optimized through the use of canine IgA, the actual target.
Prior to the described invention, it was virtually impossible to design peptides which could be used to produce antibodies of specifically targeted against canine IgA. When IgA sequences from other species are used for this purpose, the resulting antibodies have reduced affinity for the canine IgA and, therefore, reduced efficacy compared with antibodies generated using the described invention. Further, the availability of the cloned canine IgA gene enables large quantities of the canine IgA protein to be produced recombinantly for use in drug development (e.g., small molecule screening, assay development and anti-IgA antibody generation).
The DNA of the present invention may be used to identify regions of the canine IgA which are homologous to those targeted in other species and to predict novel therapeutic targets. Therapeutically interesting portions of the sequence may be expressed in chimeric proteins or used to produce peptides.
The invention also provides a renewable source of canine IgA protein through its expression using recombinant DNA techniques. This provides material for establishing assays to monitor IgA-mediated immune responses.